The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage
- University of Toronto, Canada
- Mount Sinai Hospital, Canada
- Laval University Cancer Research Center, Oncology Axis – Centre Hospitalier Universitaire de Québec Research Center – Université Laval, Hôtel-Dieu de Québec, Canada
- University of Cambridge, United Kingdom
- Princess Margret Cancer Centre, Canada
- Leiden Institute of Chemistry, Leiden University, The Netherlands
- The Hospital for Sick Children Research Institute, Canada
Figures
Figure 1 with 1 supplement
RNF168 and RNF169 bind RNF168-ubiquitylated NCPs.
(A) Schematic of RNF8-mediated DNA DSB repair pathway. ATM: Ataxia telangiectasia mutated, MDC1: mediator of DNA damage checkpoint 1, BRCT: breast cancer 1 C-terminal, H1: linker histone H1, RNF: …
Figure 1—figure supplement 1
RNF169(UDM2) binds with higher affinity than RNF168 and does not discriminate between H2AK13 and K15 ubiquitylation.
(A) Pull-down assays of NCP ubiquitylated by RNF168 using either MBP-RNF169(UDM2) or MBP-RNF168(UDM2) in the presence of varying concentrations of the acidic patch interacting KSHV LANA peptide. In …
Figure 2 with 1 supplement
Solution NMR analysis of RNF169(UDM2) reveals a flexible LRM2.
(A) Primary sequence of MIU2 (orange) and LRM2 (blue) modules of both RNF169 and RNF168. Conserved residues are indicated with vertical bars. (B) Assigned 1H-15N HSQC spectrum of 15N,13C-labeled …
Figure 2—figure supplement 1
Modified RNF169(UDM2) construct exhibits higher thermo stability.
(A) Primary sequence of the modified RNF169(UDM2) construct used for NMR experiments at 45°C; addition of four N-terminal residues shown in bold. (B) Temperature melt of wild-type RNF169(UDM2) and …
Figure 3 with 3 supplements
Thermodynamics and kinetics of the RNF169(UDM2)-H2AK13C ub-NCP interaction.
(A) Selected regions of 1H-13C HMQC spectra of ILV-methyl labeled ubiquitin in H2AK13Cub-NCPs with increasing amounts of unlabeled RNF169(UDM2). Arrows indicate direction of peak movement. Data …
Figure 3—figure supplement 1
Preparation and validation of disulfide linked H2AK13Cub-NCPs.
(A) Schematic outlining the chemical activation of H2AK13C and conjugation with ubG76C. (B) NMR derived diffusion constants for free ubiquitin (black), H2AK13Cub-NCP (red) and ¼ proteasome (green). …
Figure 3—figure supplement 2
RNF169(UDM2) binds ubiquitin through its canonical hydorphobic face in both the free and NCP-bound context.
(A) Overlay of 1H-13C HMQC spectra of ILV-methyl labeled ubiquitin with increasing amounts of RNF169(UDM2), as indicated. Data recorded at 11.7 T, 45°C. Final RNF169(UDM2) to ubiquitin ratio of …
Figure 3—figure supplement 3
Selected regions of CT-1H-13C HSQC spectra of 15N,13C wild-type RNF169(UDM2) with increasing amounts of unlabeled ubiquitin.
Arrows indicate direction of peak movement. Data collected at 11.7 T, 35°C.
Figure 4 with 1 supplement
NMR and mutagenesis identify the nucleosome acidic patch as the binding interface for RNF169(UDM2).
(A) Superimposed 1H-13C HMQC spectra of ILV-methyl labeled H2A (left panel) and ILV-methyl labeled H2B (right panel) in the context of the H2AK13Cub-NCP without (black) and with (yellow, left; red, …
Figure 4—figure supplement 1
Overlay of 1H-13C HMQC spectra of ILV-methyl labeled H3 H2AK13Cub-NCPs with (red) and without (black) RNF169(UDM2).
No significant CSPs are observed. Data recorded at 14.1 T, 45°C.
Figure 5 with 3 supplements
R689 and L699/R700 are critical to the formation of the complex.
Isoleucine region of 1H-13C HMQC spectra of ILV-methyl labeled ub H2AK13C-ubNCP without (black) and with (red) wild-type RNF169(UDM2) and the indicted RNF169(UDM2) LRM2 mutants. The ratio of …
Figure 5—figure supplement 1
Alanine scanning of LRM2 reveals criticial residues in RNF169(UDM2)-ubNCP interaction.
(A) Quantification of 53BP1 foci in U2OS cells transfected with GFP-RNF169(UDM2) wild-type or mutant, as indicated along the horizontal axis. CTRL: untransfected cells, LARA: L699A/R700A. Error bars …
Figure 5—figure supplement 2
Methyl resonances of LRM2 region are overlapped in 1H-13C CT-HSQC of 15N,13C wild-type RNF169(UDM2).
Significant resonance overlap of leucine residues is observed. Data recorded at 11.7 T, 35°C.
Figure 5—figure supplement 3
NMR reveals a similar acidic patch binding mode for R689 and R700 of RNF169(LRM2) and LANA(1-23).
(A) Alignment of the primary sequences of RNF169(UDM2) and the LANA peptide. Common residues that have been shown to be relevant for NCP binding are shaded in gray. (B) Selected regions of 1H-13C …
Figure 6
The RNF169 MIU2-ubiquitin interaction involves the canonical binding surface of ubiquitin and a central alanine in the MIU2.
(A) Overlaid regions of 1H-13C CT-HSQC spectra of 13C-labeled RNF169(UDM2) upon addition of increasing amounts of free ubiquitin. Residues with significant chemical shift changes are labeled. Data …
Figure 7 with 1 supplement
Ubiquitin is highly dynamic in the absence of RNF169(UDM2).
(A) H2AK13Cub-NCP cryo-EM map at 8.1 Å resolution and (B) RNF169(UDM2) bound H2AK13Cub-NCP cryo-EM map at 6.6 Å resolution including the drosophila NCP crystal structure (2PYO) (Clapier et al., 2008a…
Figure 7—figure supplement 1
Cryo-EM data and processing.
(A) Representative cryo-EM micrographs. Several individual particle projections are boxed. (B) Examples of 2D class average images from cryo-EM of the RNF169(UDM2)-ubNCP complex. (C) 3D …
Figure 8 with 2 supplements
Structural model of the RNF169(UDM2)-ubNCP complex.
(A) Alignment of ten representative members of the RNF169(UDM2)-ubNCP structural ensemble obtained from replica-averaged MD simulations constrained by CSPs and mutagenesis data. Histones H2A and H2B …
Figure 8—figure supplement 1
Schematic outline of replica-averaged MD protocol used.
Restraints used in each stage are indicated in boxes; COM: centre of mass, CS: chemical shifts, NOEs: Nuclear Overhauser effects, contacts: CSPs and mutagenesis restraints. Temperature and timescale …
Figure 8—figure supplement 2
Initial position of ub in RNF(UDM2)-ubNCP starting structures does not influence the final ub orientation.
(A) Alignment of RNF(UDM2)-ubNCP starting conformations for replica-averaged MD simulations. Histones H2A and H2B were used to align the structures with only one copy of the histones and DNA shown …
